Lyophilization, or the process of freeze-drying, is a commonly used technique to remove water in the preparation of dehydrated products. Generally, “freeze-drying” an aqueous composition involves three steps. First, the aqueous composition is frozen under conditions of low temperature. Secondly, the frozen water is removed by sublimation under conditions of reduced pressure and low temperature. At this stage, the composition usually contains about 15% water. Third, the residual water is further removed by desorption under conditions of reduced pressure and higher temperatures. At the end of the lyophilization process, a freeze-dried product, also called a “pastille” or “cake” is produced. The freeze-dried product contains very low residual water (from about 0.5% to about 5% weight/weight) and dry material in an amorphous form. This specific state is qualified as “vitreous.” Prior to use, the dried products must be rehydrated, which can greatly compromise the integrity of biological structures, especially in the case of whole cells/organisms. But damage to the freeze-dried products, including loss of viability, diminished immunogenicity, and impaired/abolished infectivity, can occur during any of the above-recited steps (i.e. freeze-drying, storage, reconstitution, and post-reconstitution storage). Accordingly, experimentally-determined stabilizers must be added to compositions, prior to subjecting them to lyophilization, and the magnitude and kinetics of each step, particularly the rehydration step, must be precisely defined (Mille et al. (2003) Biotech Bioeng. 83, 578). At the time of this filing, Applicants are aware of no safe and effective stabilizers or freeze-drying methods for preserving the viability, infectivity, and immunogenicity of protozoan parasites, including T. gondii, during all phases of the freeze-drying/rehydration process.
Stabilization of biological ingredients in dry form has typically involved the preservation of antitoxins, antigens and bacteria (Flosodort et al (1935) J. Immunol. 29, 389). However, a limitation in this process included partial denaturation of proteins when dried from an aqueous state at ambient temperatures. Drying from the frozen state helped reduce denaturation and led to better, although incomplete, preservation of biological ingredients including bacteria and viruses (Stamp et al. (1947) J. Gen. Microbiol. 1, 251; Rightsel et al. (1967) Cryobiology 3, 423; Crowe et al. (1971) Cryobiology 8, 251). Nevertheless freezing must be managed in order to maintain an optimal viability of such biological ingredients (Dumont et al. (2004) Appl. Env. Microbiol. 70, 268).
Immunogenic compositions and vaccine compositions comprising biological ingredients, such as viruses, bacteria, parasites, fungi, proteins, polypeptides, glycoproteins, and especially, attenuated live microorganisms, are markedly sensitive to the conditions by which they are prepared, formulated and stored. Such biological ingredients can be modified and degraded by chemical reactions (e.g. hydrolysis, deamination, Maillard's reaction), many of which are mediated by water. Liquid water allows for molecular movements and can result in modification of protein conformations in compositions comprising biological ingredients. By limiting access to water, or by removing water, a major factor of modification and degradation is reduced. This water transfer must be carefully regulated to avoid yeast damages (Gervais et al. (1992) Biotech. Bioeng. 40, 1435). Prior methods to confer stability to biological ingredients have primarily involved freezing the water or removing water by freeze-drying.
More recently, sugars such as sucrose, raffinose and trehalose have been added in various combinations as stabilizers prior to lyophilization of viruses. A large number of compounds have been tested for their ability to stabilize different vaccines containing live attenuated biological ingredients, in particular viruses. Such compounds include SPGA (sucrose, phosphate, glutamate, and albumin; Bovarnick et al. (1950) J. Bacteriol. 59, 509-522; U.S. Pat. No. 4,000,256), bovine or human serum albumin, alkali metal salts of glutamic acid, aluminum salts, sucrose, gelatin, starch, lactose, sorbitol, Tris-EDTA, casein hydrolysate, sodium and potassium lactobionate, and monometallic or dimetallic alkali metal phosphate. Other compounds include, for example, SPG-NZ amine (e.g. U.S. Pat. No. 3,783,098) and polyvinylpyrrolidone (PVP) mixtures (e.g. U.S. Pat. No. 3,915,794). To preserve live attenuated flaviviruses, one group has combined a complex mixture of multiple compounds, including sorbitol, sucrose, optionally trehalose and/or other disaccharide or trisaccharides, urea, and a specific combination of amino acids (U.S. Pat. No. 8,142,795, to Sanofi Pasteur).
As regards preserving still more complicated biological structures, one reference discloses a process for lyophilizing of a mixture of Anaplasma (a bacterium), Toxoplasma cells, and blood cells (WO 92/14360, to Cryopharm Corporation). This method keeps the parasites viable, but only in the context of their host cells. More recently, WO 2009/099075 (Snow Brand Milk Product Co., Ltd) disclosed a viability-preserving, freeze-drying method for bifidobacterium, lactobacillus, streptococcus and lactococcus, involving milk components. Even more recently, WO 2012/098358 (Biopharma Technology Ltd) described a method for freeze-drying cell-based biological material while retaining some viability after reconstitution.